Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia.

A flow cytometric approach to minimal residual disease (MRD) monitoring useful in childhood B-lineage acute lymphoblastic leukemia (ALL) is discussed here in the context of ALL in adults. Of 64 leukemia samples analyzed, 95.3% had at least one abnormal phenotype (57.3% had two or more) as compared to physiologic B-cell precursors in adult bone marrow. The method was sensitive enough to detect one leukemic cell among 10,000 normal cells in 16/19 experiments (84.2%). Blast phenotypes were stable in culture and at relapse, and were useful for MRD monitoring in patients. Marker combinations for childhood ALL are also applicable to adult cases.

Effect of addition of FLT-3 ligand and megakaryocyte growth and development factor on hemopoietic cells in serum-free conditions.

The aim of this study was to clarify the mechanisms that regulate hematopoietic cell expansion in vitro by identifying defined culture conditions. We report the results of experiments with CD34(+) cells from cord blood (CB, n = 13), bone marrow (BM, n = 4), and mobilized peripheral blood stem cells (PBSC, n = 5) using two combinations of cytokines: (A) granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), stem cell factor (SCF), erythropoietin (EPO), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (FGF-b) and (B) combination A plus FLT3 ligand (FL) and megakaryocyte growth and development factor (PEG rhMGDF). Cultures of immunoselected CD34(+) cells were performed in serum-free liquid medium without serum substitutes. The area under the curve (AUC) obtained by plotting the logarithm of the total number of viable cells, CD34(+) cells, and CFC per well, toward the week of culture was used as an index of cell expansion. With CB, a significant difference was obtained between the two combinations of cytokines with regard to the total number of viable cells, GM-CFC, and CD34(+) cells. The difference between the two combinations of cytokines obtained with BM was significant with respect to the total number of viable cells and CD34(+) cells but not for the erythroid and myeloid progenitors. When CD34(+) cells from peripheral blood stem cells (PBSC) were cultured in presence of the two combinations of cytokines, the difference in terms of AUC was not statistically significant. Our data indicate additional effects in terms of proliferation and expansion of hematopoietic cells in serum-free conditions when FL and polyethylene glycol (PEG) rhMGDF are included in culture and suggest a differential activity of these cytokines on cells from different hematopoietic sources.

Thymic epithelial cells promote survival of human T-cell acute lymphoblastic leukemia blasts: the role of interleukin-7.

BACKGROUND AND OBJECTIVES: T-cell lymphoblastic leukemia (T-ALL) cells originate within the thymus from the clonal expansion of T cell precursors. Among thymic stromal elements, epithelial cells (TEC) are known to exert a dominant inductive role in survival and maturation of normal, immature T-cells. In this study we explored the possible effect of TEC on T-ALL cell survival and analyzed the role of interleukin-7 (IL-7) within the microenvironment generated by T-ALL-TEC interactions. DESIGN AND METHODS: T-ALL blasts derived from 10 adult patients were cultured with TEC obtained from human normal thymuses. The level of blast apoptosis was measured by annexin V-propidium iodide co-staining and flow cytometry. The proliferative response of leukemic cells to interaction with TEC was evaluated by thymidine incorporation at various time intervals of culture. To assess the role of IL-7, lympho-epithelial co-cultures were carried out in the presence of anti-IL-7 or anti IL-7R blocking antibodies and the level of apoptosis of T-ALL blasts was analyzed. RESULTS: When T-ALL cells were cultured in the presence of TEC monolayers, the percentage of viable cells increased significantly and this survival was sustained with time in culture. In addition, the interaction with TEC induced a considerable proliferative response in T-ALL cells (15-fold greater than that of the control cells after 7 days of culture). The presence of IL-7 or IL-7R blocking antibodies in lympho-epithelial co-cultures consistently reduced the TEC-mediated apoptosis inhibition in T-ALL blasts (70% decrease). INTERPRETATION AND CONCLUSIONS: These results point to the role of thymic epithelium in the regulation of T blast survival. In addition, they show that interaction between IL-7 and its receptor has the major role in modulating T-ALL survival within the microenvironment generated by the T-ALL/TEC interaction.

Outcome prediction by immunophenotypic minimal residual disease detection in adult T-cell acute lymphoblastic leukaemia.

Flow-cytometric detection of minimal residual disease (MRD) identifies patients with high relapse risk in childhood acute lymphoblastic leukaemia (ALL). We studied the efficacy of this method in adult T-ALL treated with the Italian co-operative GIMEMA (Gruppo Italiano Malattie Ematologiche dell'Adulto) LAL0496 protocol. Bone marrow samples from 53 patients were taken at fixed treatment time points and MRD was analysed using a leukaemia-specific immunophenotype (cytoplasmic-CD3/nuclear-terminal desoxynucleotidyl transferase). The median follow-up was 17 months (range 3-61) and a median of 4.5 analyses/patient was performed (range 3-12). Six out of 53 (11.3%) patients were refractory to treatment, 30/53 (56.6%) relapsed and 17/53 (32.1%) remain in continuous complete remission. The probability of relapse at 2 years for MRD-positive patients at preconsolidation was 81.5%vs 38.9% for MRD-negative patients (P = 0.00078). This risk was still 54.5% for MRD-positive vs 15.8% for MRD-negative patients pre-third reinduction (P = 0.0098) and 50.0% for MRD-positive vs 16.4% for MRD-negative patients pre-sixth reinduction (P = 0.032). The relapse-predicting value of MRD did not depend on features at diagnosis such as age, sex and leucocyte count. Our data suggest that immunophenotypic MRD monitoring in the first year of treatment is a useful outcome predictor for adult T-ALL patients.